Complex biological processes, including development, differentiation, and signal transduction, involve the coordinated expression of multiple genes and proteins, as well as control of their function. The identification and quantification of multiple proteins that constitute and control a particular process is important for understanding the regulation of biological systems. Additionally, the ability to monitor the presence or absence of particular proteins, an increase or decrease in protein expression, a change in protein microheterogeneity, or a combination of these modifications may be used for early diagnosis of a wide spectrum of known diseases (1). Proteomics, the large-scale analysis of proteins, will therefore contribute greatly to our understanding of gene function in the post-genomics era.
There are several known methods for analyzing proteins in the developing field of proteomics. All of these, however, have certain disadvantages associated with them. One currently used tool for protein identification and sequencing is mass spectrometry (MS) employing matrix-assisted laser desorption/ionization (MALDI) (2,3) and electrospray ionization (ESI) (4,5). For the analysis of complex protein mixtures such as cell lysates, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is still the method of choice for separating more than thousands of proteins (6,7,8) prior to MALDI-MS or ESI-MS. Prior to mass spectrometric analysis, individual protein spots are excised from the gel, washed, in-gel reduced, S-alkylated, and in-gel digested with an excess of trypsin. Repeated washing, drying, and swelling of gel pieces are needed between each step of chemical or enzymatic reaction. Peptides are then extracted using aqueous/organic mixtures at acidic or basic conditions and prepared for peptide mass mapping or sequencing using MALDI or ESI-MS (9,10). All of these procedures are time-consuming tasks prone to sample loss and analyte dilution.
Due to its ability to provide detailed views of thousands of proteins expressed by an organism or cell type. 2-D PAGE has been a primary tool for comparative studies of proteins, for example, between normal and cancerous cells (44,45,46,47). The two dimensions of a 2-D PAGE separation are isoelectric focusing in a pH gradient and SDS-PAGE. Each protein spot provides a rough measure of isoelectric point (pI) and molecular weight of the protein within 5-10%. Extremely high resolution of 2-D PAGE for protein separation is achieved by working under denaturing conditions. Attempts to perform native 2-D electrophoresis result in 2-D protein patterns with poor reproducibility, smears, and less distinct protein spots (7). In addition, the advantages of SDS-PAGE are that virtually all proteins are soluble in SDS and the range of relative molecular mass from 10,000 to 300,000 is readily covered. 2-D PAGE has assumed a major role in “proteomics”.
However, 2-D PAGE is a relatively slow, labor intensive, and cumbersome technology. Presently, protein identification and the study of protein modifications generally involve the separate excision, proteolytic digestion, peptide extraction, and mass spectrometric analysis of each “spot” (5,9,10,11,48,49,50). Concomitant sensitivity limitations are introduced by the necessary sample handling. Other approaches include the use of thin gel for direct protein identification (51) and membrane electroblotting for protein transfer, followed by direct protein scanning or on-membrane proteolytic digestion and peptide detection using MALDI-MS (52,53,54,55). Moreover, 2-D PAGE results from different laboratories can be difficult to compare, and sensitivity is limited by the amount of a protein needed to visualize a spot, typically in the low-nanogram range for silver staining.
A recent study (11), has demonstrated a disadvantage of 2-D PAGE in that only the higher abundance proteins were identified by the 2-D PAGE-MS strategies. The results indicated that more than half of all yeast proteins with lower abundance were not amenable to be studied by current 2-D PAGE-MS approaches. This conclusion is consistent with the combined results of other yeast proteome 2-D PAGE-MS studies that have yielded a combined total of only ˜500 identified proteins. Thus, important classes of regulatory proteins involved in signal transduction and gene expression, for example, and other lower abundant proteins remain unidentified by the current 2-D PAGE-MS methodologies.
One obvious approach to increase the detection capability of low abundant proteins is to raise the protein loading from 0.5 mg to 50 mg, clearly exceeding the capacity of 2-D PAGE (11). The extracted peptides from the spots on SDS-PAGE are fractionated and analyzed using various chromatography techniques and MS/MS. Another approach will be to separate the proteins into a number of fractions before 2-D PAGE in addition to the use of a series of narrow pH range gels (12). The disadvantage of these strategies, however, is that they all require much larger amounts of protein along with many additional 2-D PAGE and chromatography separations, and therefore may be impractical for studies of small cell populations or tissue samples.
Thus, significant research efforts have been reported in the literature toward providing better linkage between 2-D PAGE and mass spectrometric analysis. For example, Ekstrom et al. (56) have combined several silicon micromachined analytical tools, including the microchip immobilized enzyme reactor, the piezoelectric microdispenser, and the high-density nanovial target plates, into an integrated platform for performing rapid protein digestion and subsequent picoliter sample preparation in a high-density format for MALDI-MS. Harrison and coworkers (57) have reported the integration of immobilized trypsin beads within a glass chip for protein digestion followed by on-chip capillary electrophoresis separations and ES-IMS detection.
Togawa et al., (77), Gombocz et al., (78,79), Doering et al., (80), Peck et al., (81), Kambara et al., (83), and Yefimov et al., (84) all disclose apparatuses for extracting protein from a gel. However, none of these disclose methods of increasing the speed of transfer, or supply methods of concentrating the transferred proteins. Liao et al., (82) describe a method for concentrating proteins, but not methods relating to 2D gel electrophoresis.
Still, the limiting factor for linking 2-D PAGE with mass spectrometric analysis lies in the effective and rapid recovery of peptides, in particular those from low-abundance proteins, from in-gel digestion as well as the extraction and the transfer of gel proteins for sequential or parallel proteolytic digestion (9,58,59). There is a need in the art for a method of rapidly recovering low abundance peptides from 2-D gel electrophoresis. This invention satisfies that need by providing apparatus and methods for performing gel extractions.